MULTIPLE BRANCH PEPTIDE CONSTRUCTIONS FOR USE AGAINST HIV

Abstract: Multiple branch peptide constructions formed from peptides derived from the V3 loop of gp120 protein of HIV-1, and including the consensus sequence GPGR preceded by 0 to 4 amino acids and succeeded by 2 to 4 amino acids, preferably not including I in the 3 amino acids immediately preceding GPGR and most preferably GPGRAF, show increased receptor affinity and prevent cell-to-cell fusion. They have a direct virostatic effect. Because they present the same peptide sequence several times, these MBPCs are able to neutralize in vitro the different steps of virus envelope/cell membrane fusion, and infected cell membrane/uninfected cell membrane fusion of several strains of HIV-1 and HIV-2. Notably, they are effective in blockading both CD4 receptors on lymphocytes and macrophages and GalCer receptors on colon epithelial cells. These results open a potential use in treatment of HIV-1 and HIV-2 infection.

Description: MULTIPLE BRANCH PEPTIDE CONSTRUCTIONS FOR USE AGAINST HIV Background of the Invention Human Immunodeficiency Virus (HIV) is presumably the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). HIV establishes a persistent infection in different cell types; many of the cells express an antigen, the CD4 receptor, as a binding site at their surface. In such cells, the first step of cell infection is represented by viral attachment with binding of the external envelope glycoprotein of HIV to the CD4 surface antigen of the cell. This binding is followed by a series of events involving virus envelope and target cell membrane fusion and internalization, through which the cell is infected. HIV infected cells may then infect other cells, forming syncytia (giant polynuclear cells). Syncytia formation between HIV-1 infected cells and uninfected CD4 + cells (cells having the CD4 receptor) involves an interaction between the CD4 receptor and the HIV surface envelope glycoprotein. This process is blocked by soluble CD4, anti-CD4 and anti-V3 antibodies. Cell fusion processes responsible for cell-to-cell spread of the virus in vivo make plasma neutralizing antibodies obsolete and lead to virus escape from the immune system already damaged by lymphocyte depletion. B lymphocytes activated by HIV produce different antibody populations. Some antibodies do not interfere with gpl20-CD4 interaction, but block membrane fusion, the process responsible for cell infection. These antibodies are principally directed against the envelope glycoprotein V3 loop. Due to the very high variability of V3 loop in different HIV-1 isolates, anti-V3 antibodies generally only neutralize the isolate against which the antibodies were produced. Therefore neutralization is limited to that isolate and is called isolate specific. These antibodies are effective by cell fusion inhibition, without any activity on cell-virus binding. Several attempts have been made to inhibit HIV infection by V3 loop-related peptides raising questions as to the efficacy of such peptides. For example, De Rossi et al. [Virology 184, 187-196 (1991)] found that some V3 peptides enhanced viral infectivity. Other synthetic peptides from the V3 loop, cyclic or non cyclic, were ineffective in the critical step of cell fusion. Thus, antagonist peptides have not been developed which are capable of blocking virus-cell fusion and cell-to-cell fusion independently of the virus isolate. More recently, several studies have demonstrated a CD4 independent route of cell infection for both HIV-1 and HIV-2, suggesting the existence of at least one alternative viral receptor. One of these putative non-CD4 HIV receptors has been recently identified on CD4 " brain derived cells and colon epithelial cells. This receptor is a neutral glycolipid, called galactosyl ceramide (GalCer). HIV infection of CD47GalCer + cells in the brain and in the intestine may account for some of the HIV-associated disorders in these organs. Moreover, the presence of GalCer on the apical side of some mucosal epithelial cells may facilitate the entry of the virus during sexual intercourse. No peptide derived from the V3 loop has yet shown ability to block the GalCer receptor. In response to some of these problems, radially branched systems using lysine skeletons in polymers have been used by J.P. Tarn [Proc. Natl. Acad. Sci. USA, 85, 5409-5413 (1988)] to develop antigens without the use of carriers. Those antigens were designed to generate vaccines against a variety of diseases. Specifically, antigens for generating vaccines against HIV infection are described by Tarn in PCT application ser. WO93/03766 and essentially include the sequence IGPGR (IUPAC convention single letter nomenclature for amino acids) and are of eleven amino acids in length to be effective in eliciting useful immune responses. Id. at page 13, lines 29-31. Such antigens are not, however, considered as potential direct therapeutic approaches to any disease, but are intended to provoke an immunogenic response in the body. Summary of the Invention The present invention includes a method and peptide construction for the therapeutic treatment of patients with HIV infections. In accordance with this method, the multiple branch peptide construction for therapeutic administration comprises a core matrix attached to from 2 to 64 peptides, each comprising the amino acid sequence GPGR preceded by from 0 to 4 amino acid residues and succeeded by from 2 to 4 amino acid residues. The Drawings Figure 1 is a proposed binding mechanism for HIV and CD4 + or CD4 " cells. Figure 2 shows the results of testing the effect of Multiple Branch Peptide Constructions (MBPCs) on the infection of human peripheral blood lymphocytes (PBLs) for three different strains of HIV, with the amount of p24 (pg/ml) or Reverse Transcriptase (RT) activity plotted against three different MBPCs, 1-none; 2- [GPGRAF] 8 -Multiple Lysine Core (MLC); 3- [RKSIHIGPGRAFYT] 4 - MLC; 4- [PP YVEPTTTQC] 4 -MLC . Figure 3 are the results of testing the MBPCs on human macrophage infection with the MBPCs plotted against the amount of p24 (ng/ml). Five different MBPCs are used: 1- none; 2- [GPGRAF] 8 -MLC; 3- [GPGRAF] 8 -MLC D ; 4- [GPG(R) D AF] 8 - MLC; 5- [RKSIHIGPGRAFYT] 4 -MLC; and 6- [PP YVEPTTTQC] 4 -MLC . Figure 4A depicts syncytia formation in infected cultures without any MBPCs. Figure 4B depicts the absence of syncytia formation in macrophages treated with [GPGRAF] 8 -MLC. Figure 5 A illustrates the effect of MBPCs on human epithelial cells infection through the GalCer receptor. Figure 5B illustrates the ability of MBPCs to block the binding of gpl20 to the GalCer receptor. Figures 6A, 6B, 7A, 7B and 7C all illustrate the relative non-reactivity of rabbit sera immunized with a short MBPC, as compared to rabbit sera immunized with a longer MBPC. Detailed Description of the Invention I Structure The present invention relates to Multiple Branch Peptide Constructions (MBPCs) which are able to inhibit HIV infection and the spread and extension of HIV infection. The MBPCs have a core matrix onto which peptides are covalently bonded. The core matrix is a dendritic polymer which is branched in nature, preferably with each of the branches thereof being identical. The core matrix is based on a core molecule which has at least two functional groups to which molecular branches having terminal functional groups are covalently bonded. Suitable core molecules include ammonia or ethylenediamine. Suitable molecular branches include acrylic ester monomers which are polymerized onto the core molecule. Such molecules may be created to present varying number of branches, depending on the number of monomers branched from the core molecule. Contemplated for use herein are MBPCs with 2 to 64 branches and preferably between 4 and 16 branches. Exemplary for use to form the core matrix is lysine. A central lysine residue is bonded to two lysine residues, each through its carboxyl group, to one of the amino groups of the central lysine residue. This provides a molecule with four amino groups, which may be the core matrix for an MBPC having four peptides. Alternatively, one can provide a molecule with eight branches by bonding four lysine residues through their carboxyl groups to one of the amino groups of the lysine residues which are attached to the central lysine. This molecule can serve as the core matrix for an MBPC having eight peptides or can alternatively receive eight lysine residues to form a core matrix for an MBPC having sixteen peptides. As may readily be seen, larger MBPCs similarly may be constructed as necessitated, e.g. having 32 branches. The C-ends of peptides are covalently bonded to each of the branches of the core matrix to form the MBPC. The peptides may be the same, which is preferred, or may be different from one another. The resulting molecule has a cluster of peptides at the surface and an interior core matrix which is not presented and is therefore not antigenic. For examples of similar structures having peptides different from those contemplated herein, see J.P. Tarn [Proc. Natl. Acad. Sci. USA, 85, 5409-5413 (1988)] and US Patent No. 5229490 to Tarn and the references cited therein. Spacers may, if desired, be included between the peptides and the core matrix. The carboxyl group of the first lysine residue may be left free, amidated, or coupled to β-alanine or another blocking compound. Peptides can include D or L-amino acid residues. D amino acids last longer in vivo because they are harder for peptidase to cut, but the L amino acids have better activity, as discussed below. Moreover, peptide analogues, synthetic constructs using the carbon skeleton of peptides but omitting the -CONH- peptide bonds, can be employed in place of peptides. Thus, it should be understood that references to peptides herein may also be taken to include peptide analogues. It is believed that peptide analogues will be more resistant to peptidase and last longer in vivo. The MBPCs of the present invention include peptides derived from the V3 loop of the surface envelope glycoprotein gpl20 of the HIV-1 virus, and include the consensus amino acid sequence GPGR (IUPAC single letter amino acid nomenclature, i.e. Gly-Pro-Gly-Arg) therefrom. Said MBPCs are effective in inhibiting HIV infections in human cell cultures of different cell types. The inhibition appears to be independent from the viral strain, and even to be effective against HIV-2 strains. They, thus, have wholly surprising therapeutic properties. It should be noted that the corresponding monomer peptides do not have any therapeutic effect. The difference is believed to occur because the core matrix causes the peptide to undergo a conformational change. Specifically and unexpectedly, the two amino acids following the GPGR in the peptide have been found to bend when the GPGR monomer is attached to the core matrix. Thus, it is preferred that there are at least two amino acid residues succeeding GPGR in the peptides attached to the core matrix. However, because if the peptide is too long, the MBPC will become antigenic, it is undesirable to have more than four amino acids following the GPGR sequence and more than four amino acids preceding said sequence. Hence, the peptide will have twelve or less amino acids. Specific MBPCs showing activity on inhibition of infection by HIV-1 and HIV-2 retroviruses include the following which are set forth using IUPAC notation for amino acids: Table 1 - Exemplary MBPCs MBPC. l: (GPGRAFY) 8 -(K) 4 -(K) 2 -K-βA-OH or (GPGRAFY) 8 -(K) 4 -(K) 2 - -βA-NH 2 MBPC.3: (GPGRAF) 8 -(K) 4 -(K) 2 -K-βA-OH or (GPGRAF) 8 -(K) 4 -(K) 2 -K- β A-NH 2 MBPC.12: (GPGRAF) 16 -(K) 8 -(K) 4 -( ) 2 -K-βA-OH or (GPGRAF) 16 -(K) 8 -(K) 4 -(K) 2 -K- β A-NH 2 The OH terminal shown above on the β-alanine indicates the carboxyl group thereof, with the amino group being attached to the carboxyl group of the lysine residue. The NH 2 terminal indicates a modification of the carboxyl group of the β-alanine, to form a carboxamide terminal; the fi -alanine is still attached by its amino group to the carboxyl group of the lysine residue. In the examples reported hereinbelow, the OH forms were used. Although the peptides should contain the consensus sequence GPGR to possess the desired therapeutic effect, the inclusion of the conformational sequence of an isoleucine before the GPGR, such as IGPGR or IXXGPGR (where X is any amino acid residue), also from the V3 loop, has been found to render the MBPC poorly effective or ineffective as a therapeutical treatment when the peptides containing the IGPGR sequence are of twelve or less amino acid residues and thus are not preferred for use herein. The sequence of an isoleucine (I) being present within three amino acids prior to GPGR is a relatively conserved one, being found in about 98% of HIV genomes, but, surprisingly, it has been discovered to be undesirable. Despite MBPCs containing the IGPGR or IXXGPGR sequence not being preferred, MBPCs with that peptide sequence have been found to block syncytia formation if the replicated peptide is longer than twelve amino acid residues. For example, one such larger MBPC which does block syncytia formation is that in which the peptide bonded to each branch of a four-branched MBPC is RKSIHIGPGRAFYT. However, such large MBPCs are undesirable because such large molecules have been shown to elicit antibodies, even at low concentrations (10 "4 M), and are recognized and inhibited by HIV infected patients' antibodies, both of which would preclude them from being used in HIV-infected patients over a long term. These MBPCs also show toxicity on cell cultures at concentrations near their effective concentrations. Moreover, the size of these MBPCs makes them very difficult and expensive to fabricate, and more sensitive to protease degradation. The invention therefore provides an MBPC comprising a core matrix to which are bonded from 2 to 64, and preferably from 4 to 32 peptides, each of which comprises the sequence GPGR preceded by from 0 to 4 amino acid residues and succeeded by from 2 to 4 amino acid residues. Preferably, the peptides do not contain a sequence of I within three amino acids before G, such as IGPGR or IXXGPGR, where X is an amino acid residue. Preferably, the peptides bonded to an 8 or 16-branched core matrix are GPGRAF. However, it is believed substitutions are possible in the AF where the substituted amino acids have the same characteristics, and may be desirable because the AF appears to be susceptible to peptidase and therefore, may be fragile in vivo. II Preparation of MBPCs The manufacture of the MBPC structure, that of a branched core with peptides attached thereto, though previously called multiple antigenic peptides (MAPs), has been known in the art. See e.g. Tarn et al, J. Immun. 148, 914-920 (1992) and Wang et al, Science, 254, 285-288 (1991). Preferably, for small quantities (under one kilogram), a solid phase method is used to obtain the MBPCs. Stepwise assembling of the peptide chains can be carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl copoly(styrene-l % divinyl benzene). The Boc/benzyl strategy may be used, including a systematic double coupling scheme with hydroxybenzotriazole active esters (Boc-amino-acid-OBt). The final cleaving from resin is effected with anhydrous hydrogen fluoride (1 hour at 0°C). The MBPC is then washed with diethyl ether and solubilized in water. After lyophilization, the MBPC may be pre-purified on a P2 or G15 type molecular filtration column, equilibrated with 0.1N acetic acid. The eluate fraction may then be recovered. The purification step is achieved by using C 8 or C 18 reversed-phase HPLC. The MBPC may be characterized by its amino acid content after acid hydrolysis (6N HC1, 115°C, 24 hours) and electrospray mass spectrometry. Ill Therapeutic Effect and Use The invention provides a method for the treatment of HIV infections in which there is administered to a patient MBPCs comprising a core matrix to which are bonded from 2 to 64, and preferably from 4 to 32, peptides, each of which comprises the sequence GPGR preceded by from 0 to 4 amino acid residues and succeeded by from 2 to 4 amino acid residues, but preferably not the sequence IGPGR or IXXGPGR, where X is an amino acid residue. The MBPCs should be administered at serum levels between 10 "3 M and 10 "7 M, but preferably at about 10 "6 M. The MBPCs made according to the present invention inhibit both HIV infection and HIV-induced cytopathic effects in vitro, and may therefore inhibit virus multiplication and the spread of the virus in the host organism. The MBPCs made according to the invention neutralize the virus envelope-cell membrane fusion step, and also the infected cell membrane-uninfected cell membrane fusion step essential for syncytia formation, either step being thought to be indispensable for cell infection, virus multiplication and the spread of virus in the host organism. The MBPCs are able to blockade the CD4 receptor present in cells such as lymphocytes and macrophages, including the 89.6 strain of HIV, apparently by attaching to the CDR3 domain of the CD4 receptor. Thus, the MBPCs of the present invention block the formation of syncytia induced by HIV-1 and HIV-2 and inhibit infection of human peripheral blood lymphocytes (PBLs) and macrophages. Such blocking does not cause the cell to lose its ability to be activated by other antigens or mitogens, that is, the functionality of the lymphocyte is preserved by the MBPCs. Given the retention of the functionality of the lymphocytes, the potential problem of artificial AIDS is avoided with the present invention. Therefore, that the MBPCs of the present invention attached to the fusion receptor, rather than the receptor for activating the T-cells, is a major advance over any previous therapy against HIV. In addition to preventing HIV infection, the MBPCs of the present invention have been shown to suppress the production of HIV in cells which have been infected prior to treatment. One totally unexpected property of the MBPCs of the invention, and especially the preferred 8 x GPGRAF and 16 x GPGRAF MBPCs, is that they have shown an ability to bind to the GalCer receptor which is a receptor for HIV. This receptor has been shown to exist in colon epithelial cells and central nervous system CD4 " cells. This binding results in MBPCs inhibiting the infection of human intestinal cells by distantly related isolates of both HIV-1 and HIV-2. Another advantage of the MBPCs of the present invention is that they have been found to be non-toxic in rodents, rabbits and monkeys at high doses and thus, when used therapeutically will not harm the patient, in contrast to many current AIDS therapies. Another advantage of the MBPCs of the present invention is that in concentrations of 10 "4 M or less they are not immunogenic, and thus, unlike all prior art attempts of using peptides derived from the V3 loop or constructions thereof, are not vaccines. The lack of immunogenicity of the MBPCs is an advantage because an immune reaction would result in antibody production which would inhibit or destroy the MBPCs. Such immunogenic MBPCs, such as the previously mentioned RKSIHIGPGRAFYT, could only be used a few times in the same individual after which it would become ineffective. A Blockage of the CD4 Receptor Experimental data demonstrates a specific inhibitory effect of the MBPCs of the present invention, especially MBPC.3 and MBPC.12, on the process of HIV infection of CD4 + cells, and a total blockage of HIV-1 and HIV-2 induced syncytia formation. MBPC concentrations with which inhibiting effects were observed in vitro are around 10 "6 M for MBPC.3 and MBPC.12, and at higher concentrations for other MBPCs. The neutralization has been observed on all the HIV strains tested, i.e. MN, Lai, NDK, and 89.6 for HIV-1 and Rod for HIV-2. In contrast, the individual peptide fragments used in the MBPCs are inactive on syncytia formation inhibition, even at very high concentrations, e.g. 5xlO "4 M. The MBPCs may thus interact with a cellular molecule, involved in a post-binding event, i.e. fusion stage, needed for cellular infection, and used by distantly related HIV-1 and HIV-2 isolates, presenting divergent V3 loops. A good candidate for this postulated V3 binding site is the CDR3 domain of the VI region of CD4, because: i) anti-CD4 antibodies specific for this region (e.g. 13B8-2) block the fusion process during HIV-1 infection, ii) CDR3-derived synthetic peptides have some inhibitory effect in fusion assays, and iii) MBPCs recognize a synthetic peptide corresponding to the CDR3 domain of CD4. The MBPCs of the present invention bind to lymphocytes without altering the binding of the viral envelope (through the CDR2 domain), while preventing HIV infecting effects. The MBPCs also bind to the soluble CD4. Therefore, MBPC binding is inhibited in the presence of soluble CD4. The effect of MBPCs of the present invention on the interaction between region-specific anti-CD4 antibodies and the CD4 receptor in its natural environment was studied. In situ binding of the anti-CDR3 monoclonal antibody (MAb) 13B8-2 to CD4 expressed on macrophages was dramatically and specifically decreased in the presence of MBPCs of the present invention. Binding of other anti-CD4 MAbs, including those recognizing the gpl20 binding site (Leu3a and OKT4a MAbs), was identical in the absence and presence of MBPCs. These data demonstrate that MBPCs bind to the CDR3 region of CD4 and thus act, at least in part, by blocking the interaction between the gpl20 V3 loop and this domain of CD4. Despite the V3 loop of HIV-1 being considered to be an important determinant in the fusion process between HIV-1 and its target cells, it has not yet been possible to exploit this property therapeutically until the present invention. The main reason is that V3 is a hypervariable region showing a high degree of diversity among HIV-1 isolates. Neutralizing anti-V3 antibodies are generally type-specific and, in the best " case, may neutralize only closely related isolates. Because the MBPCs of the present invention are targeted to cellular and not viral determinants, they by-pass the problems inherent to envelope variability. Thus, the MBPCs used in this invention, although derived from the consensus sequence of the HIV-1 V3 loop, are able to neutralize various HIV-1 strains, including the highly divergent HIV-1 (NDK), and also the unrelated HIV-2 ROD strain. Surprisingly, the MBPCs of the present invention have also been found to inhibit infection with primary HIV-1 isolates, i.e. directly collected from patients, including strains that are resistant to AZT, J-l . The following examples, which are not intended to limit the foregoing, illustrate the efficacy, utility and breadth of use of MBPCs of the present invention with regard to HIV infection, and their binding and fusion with CD4 + lymphocytes and macrophages. Example 1 - Syncytia Formation Blockage Cells were cultured in RPMI 1640 supplemented with 5 % fetal calf serum, 1 % glutamine, 1 % streptomycin-penicillin (Gibco of Irvine, Scotland) in a humidified atmosphere with 5 % CO 2 . CEM or C8166 cells were chronically infected with HIV-1 Lai or HIV-2 Rod, or HIV-1MN, respectively. Infected cells (lxlO 4 ) were incubated for 2 hours with various concentrations of MBPCs in 96-well plates. Uninfected Molt-4 cells (4xl0 4 ) were then added in 100 -"1 of culture medium, syncytia were counted after 18 hours at 37°C. Results are shown in the following Table. Table 2 - Syncytia formation MBPC MBPC Cone. HIV-1MN HIV- 1 Lai HIV-2Rod MBPC.3 5xlO '6 M 10 '6 M + + 10 "7 M + + + + + + + + MBPC.5 5xl0 "6 M 10 "6 M + + + 10 "7 M + + + + + + + + + MBPC.4 5xlO '6 M + + + + + + + + + + + 10 "6 M + + + + + + + + + + + + 10 "7 M + + + + + + + + + + + Mono.3 10 "5 M + + + + + + + + + + + + 10 "6 M + + + + + + + + + + + + 10 "7 M + + + + + + + + + + + + MBPC.4 is (IGPGRAF) 4 -(K) 2 -K-βA-OH. Mono.3 is the hexapeptide GPGRAF and MBPC.5 is the tetradecapeptide MBPC (RKSIHIGPGRAFYT) 4 -(K) 2 -K-βA-OH. MBPC.3 is as set forth above. The presence of syncytia is indicated by + signs, allocated in approximate proportion to the quantities counted. MBPC.3 exhibited an in vitro inhibiting effect on syncytia formation induced by HIV at a concentration of around 10 "6 M, and MBPC.5 exhibited an inhibiting effect at a slightly higher concentration. MBPC.12 (results not shown) gave results similar to those of MBPC.3. By contrast, the monomeric version Mono.3 is wholly inactive; this has also been confirmed for Mono.3 at the higher concentration of 10 "4 M. Example 2 - Prevention of Infection of PBLs 50 x 10 3 8E5 cells chronically infected with a RT-deficient HIV-I(IIIB) isolate, were co-cultivated with 150 x 10 3 phytohemagglutinin (PHA) stimulated human peripheral blood lymphocytes (PBLs) in the presence of the indicated peptide in 96-well plates. The MBPCs used are described by the peptides attached, and the number thereof, to a core lysine matrix, which is designated MLC. All amino acids used for this experiment were in dextro form. Chemical synthesis of MBPCs was performed by the solid phase technique, -l i¬ as described above. The peptide chains were elongated stepwise on 4-(oxymethyl)-PAM resin using optimized t-butyloxycarbonyl/benzyl chemistry. Amino acid analyses of the purified MBPCs agreed with the deduced amino acid ratios. [GPGRAF] 8 -MLC was further characterized by electrospray mass spectrometry (experimental M r = 5672 Da). The number of syncytia was determined after 24 hours of incubation in the continued presence of the peptides and the results are presented in Table 3 below. + + + indicates that the number of syncytia present in the well was similar to control untreated wells; - indicates the total absence of syncytia in the well; +. indicates the occasional presence of few syncytia in the well. Syncytia formation in this test was blocked by anti-CD4 antibodies OKT4A (anti-CDR2) and 13B8-2 (anti-CDR3), consistent with previous reports. The core lysine structures (designated "MLCs") by themselves did not induce syncytia formation in this test. Toxicity was evaluated by either MTT assay (described below) or trypan blue exclusion technique. Table 3. Inhibition of HTV-l induced cell fusion by MBPCs Concentration (M) Peptide 5xl0 "7 5xl0 "6 5xl0 "5 GPGRAF + + + + + + + + + [GPGRAF] 8 -MLC + — — [IGPGRAF] 8 -MLC + + + + — [GPGRA] 8 -MLC + + + + + + + + + [GPGR] 8 -MLC + + + + + + + + + [GPG(R) D AF] 8 -MLC + + + — Toxic [GPGRAF] 8 -MLC D + + + + + + + + + [Ac-GPGRAF] 8 -MLC + + + + + + — [RKSIHIGPGRAFYT] 4 - MLC + + + — Toxic [RKSIHKGPGRAFYT] 4 -MLC + + + — Toxic [RKSIHTGPGRAFYT] 4 -MLC + + + — Toxic RAFVTIGK + + + + + + — Under these experimental conditions, [GPGRAF] 8 -MLC at a concentration as low as 5xlO "7 M, induced a marked inhibition of syncytia formation, while the corresponding monomeric peptide GPGRAF was not active over the range of concentration used (up to 5xlO "5 M). N-terminal acetylation ([Ac-GPGRAF] 8 -MLQ, addition of an isoleucine (I) residue ([IGPGRAF] 8 -MLC) and incorporation of D-amino acids in the GPGRAF sequence resulted in a significant loss of activity. MBPCs with less than 6 residues (i.e. [GPGRA] 8 -MLC or [GPGR] 8 -MLC), as well as MBPCs with a non-relevant sequence, did not inhibit cell fusion, demonstrating that the core matrix was not involved in the biological activity. [RKSIHIGPGRAFYT] 4 -MLC was able to inhibit syncytia formation, at a concentration of 5xlO '6 M. Because the isoleucine residue preceding the GPGRAF motif is highly conserved among HIV-1 isolates, two related constructions were synthesized with a lysine or a threonine residue in place of isoleucine. No significant differences in the anti-fusion activity of these three constructions were observed. However, it should be noted that [RKSIHIGPGRAFYT] 4 -MLC and its derivatives induced some toxicity in 8E5 cells when used at a concentration of 5xl0 "5 M and are therefore undesirable for use in human therapy. Based on these results, the more potent anti-HIV MBPC appeared to be [GPGRAF] 8 -MLC. In separate tests this molecule was also able to block the fusion between HIV- 1 (LAI), HIV-l(NDK) or HIV-2(ROD) chronically infected T-lymphblastoid CEM cells and HTLV-I-transformed MT-2 cells. Example 3 - Effect of MBPCs on Lymphocyte Functionality a) Effect of MBPCs on Antigen and Mitogen-Induced Cell Proliferation Peripheral blood lymphocytes (PBL) from 3 healthy HIV sero-negative donors were isolated from heparinized blood by the Ficoll-Hypaque technique. The culture medium was RPMI 1640 supplemented with 1 % glutamine, 1 % antibiotics and 10% heat inactivated fetal calf serum. Cells (10 5 ) were incubated in the presence of MBPCs (5xlO "6 M) with or without antigen (candidine, PPD) or mitogen (PHA). Antigen or mitogen treated cells were pulsed for eight hours with 1 mCi of [ 3 H]thymidine. Then cells were harvested and [ 3 H]thymidine incorporation in DNA was counted. b) Mixed Lymphocyte Response (MLR) In this assay, peripheral blood lymphocytes from the three healthy donors (105) were incubated with 105 cells from a mixture of 10 seronegative donors in 200 μl final volume in the presence or absence of various concentrations of MBPCs in microtitre plate wells. Lymphocyte mixtures were pulsed on day 6 for 8 hours with 1 μCl of [ 3 H]thymidine. Cells were harvested and [ 3 H]thymidine incorporation into DNA was counted in a beta counter. The results are shown in Table 4 below with MBPC.5 as set forth in the First test. Table 4 - PBL Antigen and Mitogen Induced Proliferation [ 3 H]thymidine incorporation (cpm) control MBPC.3 MBPC.5 Donor 3 PHA1 203000 162000 228000 PHA2 183000 154000 188000 PPD1 5900 3900 4000 PPD2 3200 3100 2400 Candl 3900 3200 3700 Cand2 1700 1200 1100 Donor 2 PHA1 115000 87000 115000 PHA2 112000 95000 111000 PPD1 38000 24000 24000 PPD2 33000 27000 26000 Candl 28000 17000 15000 Candl 13000 11000 9000 Donor 1 PHA1 PHA2 PPD1 1200 1300 1300 PPD2 1500 3100 2100 Candl 74000 87000 80000 Cand2 79000 100000 85000 The thymidine incorporation in cells is similar in treated and control plates. If the functionality of the PBLs had been negatively effected, the incorporation of thymidine a marker of cell reproduction, would be considerably increased in MBPC treated plates. Thus, these results demonstrate that there is no proliferation of PBLs, as is seen when they have lost their ability to respond to antigens or mitogens. Example 4 - Effect of MBPCs on the Infection of Human PBLs Peripheral blood lymphocytes (PBLs) were obtained from healthy donors, stimulated with phytohemagglutinin, and cultivated in RPMI 1640 containing 10% fetal calf serum and interleukin-2 (complete medium). Samples of 6 x 10 6 cells/ml were either treated or not treated with the indicated MBPC at a concentration of 10 "5 M and exposed to HIV-1 or HIV-2 (100 TCID 50 ) for 1 hour at 37°C in the continued presence of MBPCs. After thorough washing, PBLs were cultured in complete medium with 10 "5 M of the corresponding MBPC. The state of infection was assessed by determination of RT activity (for both HIV-1 and HIV-2 isolates) and HIV-1 p24 gag measurements (Coulter kit, cut-off 10 pg/ml) in the case of HIV-1 isolates 10 days post-infection. The MBPC treatment used were as follows: 1-none; 2-[GPGRAF] 8 -MLC; 3-[RKSIHIGPGRAFYT] 4 -MLC; 4-[PPPYVEPTTTQC] 4 -MLC (a non-HIV related MBPC) (MLC indicates a core lysine structure). The results shown in Figure 2 are representative of three separate experiments. The nature of the viral isolates used was checked by PCR (HIV-1 versus HIV-2 isolates) and RIPA for discriminating between HIV- 1 (LAI) and HIV-l(NDK). [GPGRAF] 8 -MLC and [RKSIHIGPGRAFYT] 4 -MLC treatment consistently caused a delay in the production of virus progeny as assessed by the measurement of reverse transcriptase (RT) activity in the culture supernatants: at days 10 and 13 after infection, RT activity was more than 90% lower in MBPC-treated samples than control samples. Since the peptides did not interfere with the RT assay, this is consistent with a MBPC induced inhibition of viral production. Similar results were obtained (for HIV-1 isolates) when viral production was followed by determination of p24 gag concentration in the culture supernatant. [RKSIHIGPGRAFYT] 4 -MLC was less potent than [GPGRAF] 8 -MLC for inhibition of HIV-2 infection. MBPCs which did not contain a consensus sequence from the V3 loop did not inhibit infection of PBLs. Example 5 - MBPC Inhibition of Syncytia Formation in Macrophages MBPCs were also evaluated for their ability to block the infection of human primary macrophages by a highly cytopathic macrophage-tropic isolate, HIV- 1(89.6). Mononuclear cells were isolated from leukophoresis units enriched for monocytes by Ficoll-Hypaque density separation. Macrophages were purified by adherence to plastic in RPMI 1640 supplemented with 10% fetal calf serum and 5 % human AB serum. The adherent cells were cultured for 5 days in the presence of GM-CSF (1 ng/ml) and were positive for the human macrophage marker (Boehringer Mannheim, clone 25F9). 5 x 10 5 cells were treated for 45 minutes with the indicated MBPC at concentrations of 5xlO "5 M and subsequently exposed to 10,000 TCID 50 of the macrophage-tropic isolate HIV- 1(89.6). The MBPCs used were as follows: 1-none; 2-[GPGRAF] 8 -MLC D ; 4-[GPG(R) D AF] 8 -MLC; 5-[RKSIHIGPGRAFYT] 4 -MLC; 6-[PPPYVEPTTTQC] 4 -MLC, with amino acids being in L form unless noted as being in D form. After thorough washing, the cells were fed again with medium and analyzed for HIV-1 p24 gag production and syncytia formation 4 days post-infection. The results are depicted in FIG.3 with the MBPCs plotted against the amount of p24 (ng/ml). As also indicated in FIG.3 the relative number of syncytia per well (+ + + , no inhibition; + + , 50% inhibition; +_, > 95 % inhibition) correlated with the concentration of p24 gag . Macrophages depicted in FIG.4A are untreated controls and show the presence of numerous syncytia in infected cultures. Macrophages as depicted in FIG.4B at lOOx, which were pretreated with [GPGRAF] 8 -MLC, displayed very few giant cells. [RKSIHIGPGRAFYT] 4 -MLC and [GPGRAF] 8 -MLC were able to inhibit infection, as assessed by i) the large decrease of visible cytopathic effects in macrophage cultures exposed to 10,000 TCID 50 of HIV-1(89.6) in the presence of MBPCs and, ii) the measurement of HIV-1 p24 gag concentration and RT activity in the culture supernatants. Control MBPCs did not significantly affect the infection rate of macrophages by HIV-1(89.6). Interestingly, [GPGRAF] 8 -MLC D (i.e. [GPGRAF] 8 -MLC with D-amino acid residues) and [GPG(R) D AF] 8 -MLC were significantly less active than [GPGRAF] 8 -MLC on macrophage infection. B GalCer Receptor The MBPCs of the present invention, especially MBPC.3 and MBPC.12 and their derivatives, also bind to the second receptor, Galoctosyl Ceramide receptor, (see Harouse et al, Science, 253, 320-323 (1991)), and completely block the infectivity of HIV on human colon cells. The corresponding monomeric peptides are inactive on this receptor. To obtain a total blockage of the GalCer pathway of infection in intestinal cells using the GPGRAF peptide in the MBPC, the degree of polymerization of the MBPC must be at least 8. This suggests that the conformational requirement of the V3 loop for GalCer recognition is more stringent than for binding to the CDR3 domain of CD4. CDR3 is an acidic region (comprising a glutamic acid residue at position 87 which has been reported to be essential for the fusion process), which probably interacts with basic amino acids of the V3 loop by electrostatic interactions. In contrast, the galactose head group of GalCer, which is known to be involved in gpl20 binding, is neutral polar and may require a different spatial arrangement of atoms in the V3 structure for optimal interaction. Thus, it is surprising that a therapy for blocking CD4 route HIV infection would also function with the GalCer receptor. The activity of MBPCs was evaluated on this alternative infection route as follows: HT-29 cells (5xl0 5 cells) were incubated with MBPCs (5xlO _6 M) for 45 minutes and subsequently exposed to 100 TCID 50 of HIV- 1 (LAI), HIV-l(NDK) or HIV-2(ROD) for 1 hour at 37°C. The residual inoculum was inactivated by three successive trypsinations and infection was assessed by: i) direct measurement of HIV-1 p24 gag (for HIV-1 isolates) and RT activity (for HIV-1 and HIV-2 isolates) in the culture supernatants; ii) co-cultivation with human PBLs. The cells were treated with the MBPCs as follows: 1- none; 2- [GPGRAF] 8 -MLC; 3- [GPGRAF] 8 -MLC D ; 4- [GPG(R) D AF] 8 -MLC; 5- [GPGRAF] 16 -MLC; 6-[RKSIHIGPGRAFYT] 4 -MLC; and 7- [PPPYVEPTTTQC] 4 -MLC, with all amino acids in L form unless noted otherwise. The results shown in FIG.5A, which correspond to infection with HIV-l(NDK), are representative of three separate experiments and the MBPC used is indicated by the number on the x-axis. The concentration of p24 gag (pg/ml) was measured during the co-culture experiment with PBLs (+ + + , > 10 ng/ml 10 days post-infection; + + , > 1 ng/ml 13 days post-infection; +, > 1 ng/ml 16 days post-infection; -, no detectable p24 gag 1 month post-infection). Similar results were obtained with HIV- 1 (LAI) and HIV-2(ROD). In another experiment with the same MBPCs, the effect of MBPCs on the binding of gpl20 to GalCer was analyzed using a high performance thin layer chromatography (HPTLC) binding assay. MBPCs (lO^M) were preincubated with GalCer for 1 hour at room temperature. After thorough washing, the HPTLC plates were incubated with HIV-1 recombinant gpl20 (2.5 μg/ml) followed by rabbit anti-gpl20 and 125 I-goat anti-rabbit IpG. The plates were washed in PBS and exposed to Amersham hyperfilm MP for 8 hours. As shown in FIG.5B treatment of HT-29 cells with active MBPCs protected the cells from infection by HIV-1 (NDK isolate). This antiviral effect was correlated with its ability to block the binding of gpl20 to GalCer. Neither monomeric V3 peptides nor control MBPCs displayed any antiviral activity and did not inhibit gpl20 binding to GalCer. The invention therefore additionally provides an MBPC comprising a core matrix to which are bonded from 2 to 64 (preferably from 4 to 32) peptides, each derived from the V3 loop of the surface envelope glycoprotein gpl20 of the HIV-1 virus, the MBPC being capable of inhibiting the infectivity of the HIV viruses, in both CD4 + /GalCer " and CD47GalCer + cells. C. Antigenicity. Toxicity and Immunogenicitv MBPCs of the present invention are not toxic to human PBLs in vitro or in vivo to mice repeatedly injected either intraperitoneally, intravenously or subcutaneously with MBPCs at a concentration of 10 "3 M. Mice receiving repeated doses of 1 mg of MBPCs, intraperitoneally and intravenously, have not shown any adverse effects. Additionally, monkeys (macaca sylvana) injected intravenously once, then subcutaneously, every day with doses of 5 mg/kg have not shown any sign of toxic effects after thirty days. Additionally, rabbits and mice injected with [GPGRAF] 8 -MLC did not produce significant titers of anti-GPGRAF antibodies, in agreement with the concept that MBPCs with less than 10 amino acid residues in each branch of the peptide are not immunogenic at concentration of less than 10 "3 M, the intended concentrations of use. Moreover, in vitro, the PBLs retain the ability to be activated by other antigens or mitogen. 1. Antigenicity Enzyme Linked Immunosorbent Assay (ELISA) of MBPC Immunogenicitv MBPC. l, MBPC.2 and monomeric V3 consensus peptide were tested in ELISA for their immunoreactivities against HIV-1 "1" sera or HIV-1 " sera. For this test MBPC.2 is (RKSIHIGPGRAFYT) 4 -(K) 2 -K-βA-OH and MBPC. l is (GPGRAF) 8 -(K) 4 -(K) 2 -K-βA-OH. The positivity of sera was first confirmed by Western Blot analysis using NEW LAV-BLOT kit (Diagnostic Pasteur) for the detection of anti-HIV-1 antibodies in serum/plasma. Ninety six well microtiter plates were coated with 500 ng/well of peptides. After saturation and washing, 50 μl of HIV-1 + serum (1/100 dilution) were added for 2 hr at 37°C. Staining was performed with peroxidase-coupled swine anti-rabbit IpG. 30 HIV-1 positive sera and 3 HIV-1 negative sera were comparatively analyzed in ELISA assay against V3 peptide derived from the North American/European consensus (V3 Cons) sequence and V3 MBPCs. The results are set forth in Table 5 below. Of the 30 HIV-1 positive sera: 26 react strongly with V3 Cons and with MBPC.2, one reacted weakly with both peptides, one reacted selectively with MBPC.2, and two sera did not react with any of the peptides. Interestingly MBPC. l did not react with any of the HIV-1 positive sera. As compared to MBPC.2, MBPC. l has been shown to be more effective in blocking HIV-1 and HIV-2 infection and less toxic. The fact that MBPC. l is not recognized by HIV-1 positive sera may suggest that such MBPC could not interfere with the neutralizing activity of anti-V3 antibodies. Therefore they could act in synergy to neutralize HIV-1 infection. TABLE 5 EUSA Western Blot V3 500 ng of peptide/wall Serum No. HIV-1 Consensus MBPC2 BPC1 1 + + + + + + + + + +. + - 2 + + ■ + + + + + + + + + - 3 + + + + + + + + + + + - 4 + + + + + + • + + + + + - 5 + + + + + + + + + + + - 6 + + + + + + + + + + + - 7 + + + ■ + + + ■ + • + + + + - 8 + + + + + + + •+ + + + - 9 + + + + + • + + + + + + - 10 + + + + + + + + + + + - 1 1 + + + + + + + + + + + - 12 + + + + + + + • + • + + + - 13 + + + + + + + + + + + - 14 + + + + + + 15 + • + + + + ■ + + + + • + + - 16 + + + + + + + + + + ■ + • - 17 + + + + + + + + + + + - 18 + + + + + + + + + + + - 19 + + + • + • + • + + + + + + - 20 + - - - 21 + + + - 22 + - - - 23 + - + - 24 + + + + + + + + + + + - 25 + + + + + + + + + + + - 26 + + + + + + + + + + + - 27 + + + + + + + + + + + - 28 + + + + + + + + + + + - 29 + • + + + + + + • + + + ■ + - 30 + + + + + + + + + + + - 31 - - - - 32 - - - - 33 - - - - 2. Toxicity An MTT assay, a colorimetric cell viability assay using 3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide, was used to assess the effect of MBPCs on cell viability. CEM or C8166 cells (5xl0 4 ) were cultured continuously (15 days) in the presence of various concentrations of MBPCs in the wells of 96-well microtiter plates in 200 μl final volume of RPMI 1640 medium supplemented with 5 % fetal calf serum, 1 % glutamine, 1 % streptomycin-penicillin (available from Gibco of Irvine, Scotland) in a humidified atmosphere with 5 % CO 2 . Every 3 to 4 days, an MTT assay was performed on 100 μl of cells suspension and MBPC were added to cell culture and the volume completed to 200 μl. [GPGRAF] 8 -MLC, at concentrations of 10 "7 to lO^M, was not toxic to cells in this test. Other results are shown in Table 3 above. As indicated the tetradecapeptide MLC which includes the IGPGR sequence is toxic and therefore not appropriate for therapeutic use as contemplated herein. 3. Antigenicity - Immunogenicitv Studies on MBPCs Four C57/BL6 black mice were injected daily for 36 days with 250 μl of MBPC.3 at 4 mg/ml (1 mg). The injection route was intraperitoneal. The blood samples were collected by sub-orbital eye punction. The blood samples were left for 1 hour at 37°C, then overnight at 4°C, and the supernatants (serum samples) thus obtained were separated from the pellets. Sera were tested by ELISA, looking for specific anti-MBPC.3 antibody. Additionally, two New Zealand rabbits were immunized with [GPGRAF] 8 -MLC: 400 μg of [GPGRAF] 8 -MLC in 1 ml of PBS, pH 7.4, were mixed with an equal volume of complete Freund's adjuvant and injected intradermally to each rabbit on day 0. This procedure was repeated subcutaneously with incomplete Freud's adjuvant on days 30, 60, 90 and 120. Sera were tested by ELISA, looking for specific anti-MBPC.3 antibodies. Data presented here is from the last serum samples (120 + 7 days). 96-well microtiter plates (Nunc, Rotskild, Denmark) were coated for 2 h at 37°C with various concentrations of [GPGRAF] 8 -MLC (500 to 25 ng of [GPGRAF] 8 -MLC in 50 μl of PBS), pH 7.4, per well. After saturation with 400 μl of casein (5 g/100 ml), different serum dilutions were added for 2 hours at 37°C. The controls were sera from non-immunized rabbits and mice. After rinsing (5x), 50 μl of 1:5000 of peroxidase-labelled swine anti-rabbit IgG or anti-mouse IgG (Dakopatts, Copenhagen, Denmark) were added for 1 hour at 37°C. After further rinsing (5x), 100 μl of ortho-phenylenediamine were added for 30 min in the dark at room temperature. The reaction was stopped by adding 50 μl of 4 N sulfuric acid, and optical density (OD) ratios were determined at 492/620 nm. Tables 6-10 below set forth the results, with Table 6 setting forth a summary of the results achieved with the rabbits using optical density ratios (xlO 3 ). Mo is the GPGRAF monomer at 10 μg/ml. Table 6 serum dilution 1:10 1:100 1: 1000 [GPGRAF] 8 -MLC (μg/ml) 10 rabbit A +++ + rabbit B + 5 rabbit A +++ rabbit B + 1 rabbit A +++ rabbit B + 0.5 rabbit A + rabbit B -, optical density (OD) <0.2; +, OD=0.2-0.4; ++, OD=0.4-0.8; +++, OD >0.8 Table 7 - microtiter plate n° 1 S 10 S 10 S5 S5 S 1 S 1 S.5 S.5 Mo Mo M10-2 1162 1199 1045 933 444 419 382 381 1224 1135 M 10-3 339 291 285 236 126 111 128 135 310 287 M10-4 172 138 121 132 50 51 56 38 104 185 Mctrl 622 550 527 551 337 313 360 341 516 591 Rctrl 1062 1093 986 976 631 662 621 616 945 989 R10-2 2000 2000 2000 2000 1280 1267 968 981 2000 2000 R10-3 2000 2000 2000 2000 346 349 318 296 2000 2000 R10-4 924 823 796 809 182 193 145 140 677 725 Legend: S 10 = 10 μg MBPC.3/ml; S 5 = 5 μg MBPC.3/ml S 1 = 1 μg MBPC.3/ml; S.5 = 0.5 μg MBPC.3/ml Mo = GRGRAF monomer, 10 μg/ml M 10-2 = [1-Cr 2 ] Mouse; R 10-2 = [10 "2 ] Rabbit M Ctrl = [10 "2 ] non immun. mouse; R Ctrl = [10 "1 ] non immun. rabbit Table 8 - microtiter plate n° 2 S 10 S 10 S5 S5 S 1 S 1 S.5 S.5 Mo Mo M10-2 1540 1457 1514 1344 381 429 315 841 1539 1575 M 10-3 334 291 263 273 54 65 47 82 342 335 M 10-4 132 188 88 97 15 17 13 83 95 126 Mctrl 490 464 385 231 241 289 235 851 482 500 Rctrl 437 429 386 345 214 185 117 153 376 395 R10-2 2000 2000 2000 2000 1922 87 34 716 2000 2000 R10-3 2000 2000 2000 2000 394 87 197 181 2000 1909 R10-4 734 689 678 626 113 84 80 74 588 625 Legend: S 10 = 10 μg MBPC.3/ml; S 5 = 5 μg MBPC.3/ml S 1 = 1 μg MBPC.3/ml; S.5 = 0.5 μg MBPC.3/ml Mo = GRGRAF monomer, 10 μg/ml M 10-2 = [10 "2 ] Mouse; R 10-2 = [10 "2 ] Rabbit M Ctrl = [10 "2 ] non immun. mouse; R Ctrl = [10 "1 ] non immun. rabbit Table 9 - microtiter plate n° 3 S 10 S10 S5 S5 SI S 1 S.5 S.5 Mo Mo M10-2 639 664 636 630 241 200 204 198 M10-3 172 168 159 163 50 53 48 49 M 10-4 85 84 83 84 22 22 22 21 Mctrl 513 492 486 459 257 249 286 261 Rctrl 833 238 222 213 92 92 91 88 R10-2 818 953 857 868 402 401 416 287 R10-3 342 328 323 314 168 150 148 155 R10-4 191 184 170 169 72 89 64 69 Legend: S 10 = 10 μg MBPC.3/ml; S 5 = 5 μg MBPC.3/ml S 1 = 1 μg MBPC.3/ml; S.5 = 0.5 μg MBPC.3/ml Mo = GRGRAF monomer, 10 μg/ml M 10-2 = [lCr 2 ] Mouse; R 10-2 = [10 "2 ] Rabbit M Ctrl = [10 '2 ] non immun. mouse; R Ctrl = [10 "1 ] non immun. rabbit Table 10 - microtiter plate n° 4 S 10 S 10 S5 S5 S 1 S 1 S.5 S.5 Mo Mo M 10-2 1454 1340 1233 1336 553 439 495 468 1231 1317 M 10-3 334 318 299 297 83 88 90 95 380 386 M10-4 133 113 124 103 27 21 28 28 123 122 Mctrl 453 381 349 400 215 235 275 278 468 458 Rctrl 225 203 285 288 95 86 82 86 283 185 R10-2 849 843 749 792 419 447 424 425 811 811 R10-3 332 314 310 304 221 151 163 154 245 194 RABU 263 279 255 251 105 101 134 128 214 218 Legend: S 10 = 10 μg MBPC.3/ml; S 5 = 5 μg MBPC.3/ml S 1 = 1 μg MBPC.3/ml; S.5 = 0.5 μg MBPC.3/ml Mo = GRGRAF monomer, 10 μg/ml M 10-2 = [10 "2 ] Mouse; R 10-2 = [10 "2 ] Rabbit M Ctrl = [10 "2 ] non immun. mouse; R Ctrl = [10 "1 ] non immun. rabbit R ABU = [10-2] rabbit, with peptide Abu (non-related amino acid) In view of the high amount of background reactivity observed in some of the results, further testing was undertaken to verify the non-reactivity of sera immunized with MBPC. l. Sera from rabbits immunized with either MBPC.l or MBPC.2 were tested in ELISA for their ability to bind MBPC.l, MBPC.2, Mono.l (the monomeric form of MBPC. l) and the North American/European consensus V3 sequence (V3 Cons, or Cs). Figure 6A shows that sera from rabbits immunized with MBPC.2 recognized significantly MBPC.2 and V3 Cons, while no reactivity was observed against MBPC.1 or the corresponding monomeric peptides. Figure 6B indicates that sera from rabbits immunized with MBPC. l did not react against MBPC. l (except for a weak reactivity of the serum from rabbit A), MBPC.2, V3 Cons or Mono. l. Preimmune sera used as negative control likewise did not react against either MBPC, V3 Cons or Mono.l. In another set of experiments, sera from rabbits immunized with either MBPC. l or MBPC.2 were tested (at 1/100 and 1/1000 dilutions) against various concentrations of MBPC. l, MBPC.2 and V3 Cons. Figures 7A, 7B and 7C clearly indicate that sera from rabbits immunized with MBPC.2 reacted in a dose dependant manner against MBPC.2 and V3 Cons. No reactivity against MBPC. l was detected. In contrast, sera from rabbits immunized with MBPC.1 did not react against MBPC.2, while reacting weakly against MBPC. l (note the weak reactivity of the serum from rabbit A against MBPC.l, with no reactivity of serum from rabbit B). Interestingly, these latter sera did not react with the monomeric V3 Cons, a 34-amino acid linear epitope mimicking the consensus sequence as present on gpl20. These results indicate that MBPC. l, a short V3 MBPC, did not induce a significant antibody response in rabbits, while MBPC.2, the longest V3 peptide construct, did invoke such a response. These observations are in line with the results on the immunoreactivity of MBPC. l with HIV-1 infected patients' sera. In mice, repeatedly injected at 10 "3 M, very weak titers of antibodies could be occasionally detected in sera at 1:100 dilution, and essentially no antibodies could be detected at lower serum dilutions. In rabbits, antibodies are detectable in one animal at 1: 100 serum dilution. At all other serum dilutions, the antibodies titers are comparable to those obtained in control animals. Such weak antigenicity demonstrates that the MBPCs of the present invention do not have the potential to be used as an antigen able to elicit an effective antibody response, which is the use of previously known peptide constructions. Indeed, the intended therapeutic concentrations of the MBPCs of the present invention are around lO^M, and no antibody response is expected at such concentrations. However, given the lack of antigenicity the MBPCs may be administered at up to 10 "3 M.

Claims: CLAIMS 1. A multiple branch peptide construction comprising a core matrix to which are bonded from 2 to 64 peptides, each of which comprises the amino acid sequence GPGR preceded by 0 to 4 amino acid residues and succeeded by 2 to 4 amino acid residues, but essentially free of the amino acid sequence IGPGR or IXXGPGR, where X is an amino acid residue. 2. A peptide construction according to claim 1 in which there are from 4 to 32 peptides bonded to the core matrix. 3. A peptide construction according to claim 1 or claim 2 in which each peptide is the same. 4. A peptide construction according to claim 1 or claim 2 in which each peptide is GPGRAF. 5. A peptide construction according to claim 4 in which there are 8 or 16 peptides GPGRAF. 6. A peptide construction according to any preceding claim in which the core matrix is comprised of lysine residues. 7. A peptide construction according to any preceding claim in which there are spacers between the core matrix and the peptides. 8. A peptide construction according to claim 1 in which the peptides are peptide analogues. 9. A peptide construction according to claim 1 in which the peptides or some of the peptides include one or more amino acid residues in dextro form. 10. A multiple branch peptide construction comprising a core matrix to which are bonded from 2 to 64 peptides each derived from the V3 loop of the surface envelope glycoprotein gpl20 of the HIV virus, the multiple branch peptide construction being non-immunogenic at serum concentrations less than 10 "4 molar and being capable of inhibiting the infectivity of the HIV virus in both are bonded CD4 + /GalCer " and CD47GalCer + cells. 11. A medicament comprising a multiple branch peptide construction according to any preceding claim in admixture with a pharmaceutically acceptable diluent or carrier. 12. Use of a multiple branch peptide construction comprising a core matrix to which are bonded from 2 to 64 peptides, each of which comprises the amino acid sequence GPGR preceded by 0 to 4 amino acid residues and succeeded by 2 to 4 amino acid residues, for the preparation of a medicament for the treatment of HIV infections. 13. A method of treating HIV infections comprising administering to a patient a multiple branch peptide construction comprising a core matrix to which are bonded from 2 to 64 peptides, each of which comprises the amino acid sequence GPGR preceded by 0 to 4 amino acid residues and succeeded by 2 to 4 amino acid residues. 14. A method of treating HIV infections comprising administering to a patient a multiple branch peptide construction according to any of claims 1 to 10. 15. A method of treating HIV infections comprising administering to a patient a medicament according to claim 11. 16. A method for the preparation of a multiple branch peptide construction comprising a core matrix to which are bonded from 2 to 64 peptides, each of which comprises the amino acid sequence GPGR preceded by 0 to 4 amino acid residues and succeeded by 2 to 4 amino acid residues, but essentially free of the amino acid sequence IGPGR or IXXGPGR, where X is an amino acid residue, the method comprising solid phase stepwise elongation of the peptide chains on a resin, followed by cleavage of the multiple branch peptide construction from the resin. 17. A method according to claim 16 in which the resin is 4-oxymethyl-phenylacetamidomethyl copoly(styrene-l % divinylbenzene). 18. A method according to claim 16 or claim 17 in which the Boc/benzyl strategy is used, including systematic double coupling with hydroxybenzotriazole active esters (Boc-amino-acid-OBt). 19. A method according to any of claims 16 to 18 in which the cleavage from the resin is effected with anhydrous hydrogen fluoride at 0°C. 20. A method according to any of claims 16 to 19 in which the prepared peptide construction is one in which there are from 4 to 32 peptides bonded to the core matrix. 21. A method according to any of claims 16 to 20 in which the prepared peptide construction is one in which each peptide is the same. 22. A method according to any of claims 16 to 21 in which the prepared peptide construction is one in which each peptide is GPGRAF. 23. A method according to claim 22 in which the prepared peptide construction is one in which there are 8 or 16 peptides GPGRAF. 24. A method according to any of claims 16 to 23 in which the prepared peptide construction is one in which the core matrix is comprised of lysine residues. 25. A method according to any of claims 16 to 24 in which the prepared peptide construction is one in which there are spacers between the core matrix and the peptides. 26. A method according to any of claims 16 to 19 in which the prepared peptide construction is one in which the peptides are peptide analogues. 27. A method according to any of claims 16 to 19 in which the prepared peptide construction is one in which the peptides or some of the peptides include one or more amino acid residues in dextro form.

Inventors: BENJOUAD ABDELAZIZ, FENOUILLET EMMANUEL, GLUCKMAN JEAN-CLAUDE, MABROUK KAMEL, ROCHAT HERVE, SABATIER JEAN MARC, VAN RIETSCHOTEN JURPHAAS, YAHI NOUARA, BENJOUAD, ABDELAZIZ, FENOUILLET, EMMANUEL, GLUCKMAN, JEAN-CLAUDE, MABROUK, KAMEL, ROCHAT, HERVE, SABATIER, JEAN, MARC, VAN RIETSCHOTEN, JURPHAAS, YAHI, NOUARA
Applicants: ARMEL SA, MCKELVEY IAN EDWARD, ARMEL S.A., MCKELVEY, IAN, EDWARD
Application year: 1994
Main classification: C07K14
IPC codes: A61K38, C07K14